The invention relates to a method of producing neural cultures and particularly, but not exclusively, neural cell-lines; and the cells and cell-lines produced by such a method.
The invention also relates to human and animal neural cell-lines, and particularly but not exclusively, nerve cell-lines.
Nerve cells are highly differentiated cells comprising a cell body, and processes, the latter subdivided into dendrites and axons. Nerve cells vary considerably in shape and size in different parts of the body. For example, granule cells from the cerebellum are 5 micrometres in diameter whereas the large motor cells of the anterior horn of the spinal cord are up to a 120 micrometres in diameter. In addition, the axons of nerve cells vary from about a hundred micrometres in length up to 1 metre in length. In addition to this variation in shape and size nerve cells also vary in the nature of the receptors expressed on their cell surface and the nature of the neurotransmitters secreted for the purpose of effecting nerve cell transmission. This difference in biochemistry can be used for the purposes of classification. Thus, in simplistic terms, nerve cells may be classified as, for example, adrenergic, cholinergic, serotoninergic, dopaminergic etc according to the nature of their neurotransmitters. This biochemical mode of classification can be further sub divided in order to identify a whole range of nerve cells secreting different neuropeptides that are thought to function as neurotransmitters or neuromodulators such as the neuropeptides beta endorphin, metxe2x80x94enkephalin, somatostatin, luteinizing hormonexe2x80x94releasing hormone, thyrotropin releasing hormone, substance P, neurotensin, angiotensin 1, angiotensin 2, vasoactive intestinal peptide, neuropeptide Y, calcitonin gene related peptide etc, or alternatively, the amines or amino acids, adrenalin, noradrenaline, octopamine, serotonin, histamine, gamma aminobutyric acid, and taurine. The afore list is not intended to be exhaustive but rather serves to illustrate the nature of the biochemical diversity of nerve cells.
It is widely acknowledged that it would be immensely advantageous if it was possible to provide ideally in culture a homogeneous population of nerve cells and so provide, for example, a homogeneous population of nerve cells either from a given location in the central nervous system, or alternatively, a homogeneous population of nerve cells exhibiting either predetermined morphological characteristics and/or biochemical characteristics. For instance it would be highly advantageous if it was possible to provide a homogeneous population of nerve cells which were characterised by either the transmitter secreted in response to activation or alternatively the receptor occupied in response to activation. With such a population of nerve cells it would be possible for research biologists to make significant advances in the understanding of the nervous system and for industrial biologists to manufacture and test drugs, agents or entities which affect the functioning of a given population of nerve cells with a view to developing therapeutically active agents.
In addition, if it was possible to provide a homogeneous population of nerve cells it would be possible to provide nerve cells of a given classification for the purpose of transplantation. This would be particularly appropriate in cases where nerve cell degeneration or damage had occurred. For example, it is well known that Parkinson""s Disease is related to nerve cell degeneration and a corresponding lack of secretion of dopamine by nerve cells. Thus, if it was possible to provide a homogeneous population of nerve cells that secrete dopamine then it would be possible to transplant such nerve cells and thus mitigate or alleviate or even reverse the symptoms of Parkinson""s Disease. Similarly, other forms of dementia which are characterised by a progressive degeneration of nerve cells could be treated in a similar manner. Similarly, acute destruction of nervous tissue could be treated by nerve cell implants comprising a homogeneous population of nerve cells and/or the implantation of a selected combination of nerve cells from different homogeneous populations.
However, the above referred to diversity of nerve cells and also the postmitotic nature of nerve cells tend to impose severe restrictions on the number of cells that can be obtained in vitro for investigation and/or transplantation using conventional cell culture techniques. For this reason attempts have been made to provide cultures of nerve cells by cultivating primary tumour tissue or by fusing primary cells with tumour cells. However, tumour cells are irreversibly transformed and have an ill-defined history. Their use as cell models is therefore highly questionable and moreover because of the potential tumorigenicity of such tissue they cannot be used for the purpose of transplantation.
Attempts to provide homogeneous populations of nerve cells have also been undertaken using carcinogen-induced transformation both in vivo and in vitro and also by spontaneous transformation that is to say by the out growth of cells from primary cultures without any deliberate genetic manipulations.
However, it has been found that another restriction on the provision of homogeneous populations of nerve cells concerns the fact that most neural tumours are human glioblastomas and thus do not concern the uncontrolled division of functional nerve cells.
Other workers have transfected neural cells with oncogenes in order to establish neural cell-lines. Some workers have shown that it is possible to induce oncogenes into primary neural cells and to obtain cell-lines, however, these cell-lines are not nerve cell-lines. They are not functioning nerve cells nor are they homogeneous populations of definable nerve cells (1).
The transfection techniques used in the past have involved the use of retroviruses because of the ability of such viruses to stably integrate into the host cell genome. In addition, transfection has been undertaken using a temperature sensitive mutant of the DNA virus simian virus 40 (SV40). The A gene of SV40 encodes the large tumour (T) antigen which is required for the initiation and maintenance of transformation.
Integration of viral genes into host cell genomes requires that the host cell undergoes at least one round of DNA synthesis. It therefore follows that where integration of a viral gene into a host cell is required target cells are limited to mitotic neural cells. Transfection techniques have therefore been undertaken on such cells. Although it has been possible to produce cell-lines, that is to say it has been possible to immortalise the transfected cells, it has not been possible to produce immortalised cells with the required degree of differentiation which would render such cells as useful tools for further research, study or use. This would seem to be because immortalisation prevents terminal differentiation of nerve cells. Indeed, typically the cells enter crisis and apoptosis ensues. For example, when immortalisation of neural cells takes places using SV40 T a homogeneous population of cells can be cultured, however at a non permissive temperature of 39xc2x0 C. expression of the active viral protein ceases and the cells enter differentiation. However, differentiation does not proceed to completion, the cells enter crisis and apoptosis ensues.
In addition, it also widely acknowledged that it is extremely difficult to provide in culture differentiated neural or nerve cells either for use in transplantation and/or for use in testing drugs, agents or entities which effect the functioning of a given population of nerve cells with a view to developing therapeutically active agents. It is difficult to provide such a culture of nerve cells, especially where one is trying to provide, largely, a homogeneous population of nerve cells, or a heterogeneous population of nerve cells including a relatively small number of phenotypes, because, amongst other things, it is very difficult to provide for differentiation of such nerve cells. Typically it is difficult to provide for differentiation of primary nerve cells in culture.
It is therefore an object of the invention to provide a method for producing nerve cell-lines which represent homogeneous populations of nerve cells which are not only functional but whose character can be reliably defined. In other words it is an object of the invention to provide a method for producing a stable nerve cell-line which is committed to its phenotype. For example, using the invention it is possible to provide a homogeneous population of functional serotonin cells or acetylcholine cells or adrenalin cells etc.
It is a further object of the invention to provide a non-mitotic cell-line, whose non-mitotic characteristics persist even in the presence of factors and/or conditions which would normally promote mitosis.
It is yet a further object of the invention to provide a cell-line which survives at low densities.
It is also an object of the invention to provide a method for producing nerve cell-lines which can be selectively made to enter apoptosis so that the process of apoptosis can be studied with a view to gaining a greater understanding of the process and also with a view to engineering drugs, agents or entities that affect apoptosis.
It is yet a further object of the invention to provide for a population of nerve cells, homogeneous or otherwise which are fully differentiated.
The method of the invention is based on a startling observation. Using conventional transfection techniques we were able to immortalise selected neuronal cells. However, as with many other workers, until realising the invention, we were unable to provide fully functional differentiated nerve cells. However, when we modified our method for producing cell-lines we found that we were able to induce full differentiation of our nerve cells when they were exposed, following transfection and immortalisation, to predetermined conditions. These conditions involved exposing the cells to either the environment from which they came and particularly, but not exclusively, the mitotic environment from which they came or to conditions which mimicked the environment from which they came and thus provided for an artificial imitation of the environment from which they came.
Our observation has also enabled us to produce an in vitro culture of nerve cells which have not been immortalised. In this instance, primary nerve tissue is first encouraged to replicate by exposure to a replicating agent (8 and 9) and is then encouraged to differentiate by exposing the cell culture to the aforementioned environment from which said primary tissue came or to conditions which mimic said environment.
By the term, the environment from which they came, we mean a region of the central nervous system, and more preferably a region of the central nervous system at, adjacent, or functionally related to the natural location in the central nervous system of the cultured cells. We favour a mitotic environment therefore we favour a region from the central nervous system which is mitotically active and more preferably we favour a region from the central nervous system at, adjacent, or functionally related to the natural location in the central nervous systems of the cultured cells.
It would seem that having to expose the cells to the environment from which they were derived means that cells of that environment secrete agents, such as for example cytokines, growth factors, transmitters etc or perhaps such cells comprise removable cell surface based factors, which can elicit a differentiation response.
In addition, we have found that it is possible to use tissue and cells from different species in order to work the invention. For example, it is possible to culture human nerve cells and expose such human nerve cells to said environment or said artificial environment which is derived from rat central nervous system. Conversely, it is possible to culture rat nerve cells and expose said nerve cells to an environment or artificial environment which is human derived.
It would therefore seem that agents which elicit neuronal differentiation of the invention are agents which can elicit their effects cross species. That is to say these agents are biologically active in at least both rat and human systems and are therefore likely to be of the same or similar structure.
Thus we have found that modifying our method such than transfected cells or cultured cells are exposed to the conditions of the original environment at least from which the first culture cell came brings about differentiation. We are unclear as to the nature of the factors involved at this stage.
Further, when using transfected cells we prefer to employ a method which includes the provision of a switch which enables us to control immortalisation and apoptosis. Using our method we have found that cultured nerve cells do not spontaneously undergo apoptosis so frustratingly characteristic of previously cultured neural cell-lines, but rather we can selectively control whether cell-lines remain immortalised or enter apoptosis.
In addition, we have also found that our cell-lines when differentiated, are committed to their phenotype and thus retain their phenotypic characteristics even when the environment from which they came is removed and/or they are exposed to factors such as foetal calf serum. Further, we have also found that our cell-lines do not exhibit mitosis, again, even under conditions which would promote mitosis, and, what is more, our cell-lines are able to survive at low densities.
According to a first aspect of the invention there is therefore provided a method for producing large populations of neural cells which method involves:
a) enhancing the replication of a first undifferentiated neural cell, or neural cell precursor cell, or precursor stem cell,
b) exposing said replicated neural cells either to an environment from which said first neural cell came, or to an environment which mimics said environment; and
c) allowing differentiation of said cells to produce fully differentiated active neural cells.
It is apparent from the above that using the method of the invention one is able to culture and/or immortalise a neural cell precursor cell and thus produce a homogeneous population of cells. However, successful differentiation is effected by exposing the cells to either the environment from which the first nerve cell came or alternatively to an environment which mimics that environment. In this way, it is possible to produce a homogeneous population of fully differentiated active neural cells.
In a first embodiment of the invention the environment from which the first nerve cell came is any region of the central nervous system, however, more preferably, said environment is an environment at, adjacent, or functionally related to the natural location in the central nervous system from which the cultured cells derive. The term, an environment which mimics said environment, is also to be construed accordingly.
More preferably still, said environment is a mitotic environment, that is to say, it comprises cells undergoing mitosis. It would seem that in this instance the agent(s) which elicit the differentiation process are being released or expressed and somehow affecting differentiation by cells within the mitotic cells environment.
Preferably, said nerve cells and tissue from said natural or artificial environment is derived from a single species. However, alternatively, said nerve cells and said tissue may be derived from different species. For example, said nerve cells may be derived from foetal human tissue whereas said environment and more specifically said tissue of said environment may be derived from an another animal species such as rat, mice, monkeys etc.
In a preferred embodiment of the invention immortalisation is achieved by using conventional transfection techniques and preferably the transfection involves the incorporation into the cell genome of an oncogene which oncogene favours the establishment of cell division well beyond the normal level encountered when a cell is not transduced with an oncogene, in other words the oncogene immortalises the cell.
Alternatively, immortalisation may be effected using physical or chemical means. For example, immortalisation may be effected by exposing said cell to radiation or chemicals (2) which are known to promote cell division well beyond the normal level encountered when a cell is not exposed to said physical and chemical means.
Ideally transfection is undertaken using a virally derived oncogene such as a myc, src, ras, SV40T, or a retroviral construct including any of the aforementioned oncogenes and/or any human oncogenes. A retroviral construct is favoured because of its ability to stably integrate into the host cell genome.
In a first preferred embodiment of the invention the immortalising agent includes or has associated therewith a control means whereby activation of the control means terminates immortalisation and causes the cell to enter apoptosis.
It is preferred that immortalisation of said cell with an immortalising agent takes place ideally during the last division before migration from the proliferative zone and the onset of terminal differentiation. This is because the likelihood of producing a cell-line having a single set of functional characteristics is increased. Immortalisation prior to this preferred time can be undertaken but the likelihood of the precursor cells adopting several different phenotypes after differentiation is increased.
In a preferred embodiment of the invention the control means is responsive to culture or environmental conditions such as temperature, pH or ionic concentrations. For example, in a preferred embodiment the immortalising agent is temperature sensitive and the control is thus represented by a temperature sensitive switch so that at, about, or below a first given temperature the immortalisation agent is activated so as to immortalise the selected nerve type, but at, about, or above a second temperature the immortalising agent is deactivated and in this instance immortalisation terminates and apoptosis is allowed to proceed. The immortalisation agent and the control means may comprise, for example, a single entity such as a temperature sensitive oncogene. Alternatively, the immortalisation agent and the control means may be two independent entities but in either case ideally the activation/deactivation of the control means has a reciprocal effect on the immortalisation agent. For example, when the control means is activated the immortalisation agent is deactivated. Conversely when the control means is deactivated the immortalisation agent is activated. This ability of the control means to deactivate the immortalisation agent is a means of terminating immortalisation such that apoptosis can take place.
Exposing said cells to the original environment can involve transplanting said homogeneous population of cells back into the central nervous system or more preferably a location in the central nervous system at, adjacent or functionally related to the original environment of the first cell or alternatively, and more preferably, simply extracting a population of cells from said central nervous system or said original environment and placing said extracted population in close proximity to said homogeneous population of cells.
Ideally, said chosen environment comprises mitotically active cells.
In the instance where said cell is exposed to an extracted population of cells then ideally said extracted cells are plated onto a substrate and allowed to reach confluence either before being placed in contact with said homogeneous population of cells or whilst in contact with said homogeneous population of cells. Alternatively, said extracted population of cells are grown to confluence and medium from said population is added to said homogeneous population of cells in order to bring about differentiation.
Preferably, said homogeneous population of cells are also exposed to one or more growth factors such as fibroblast growth factor and/or epidermal growth factor.
It will be apparent from the above that the nature of the homogeneous population of cells will be determined by the nature of the undifferentiated nerve cell or nerve cell precursor cell. Thus using the method of the invention it will be possible to produce cell-lines of different nerve cells whose function and properties will be determined by the nature of the undifferentiated nerve cells or nerve cell precursor cells. Thus the invention has wide ranging application in that the invention provides a method whereby a whole range of homogeneous populations of nerve cells can be grown in culture. This is obviously significant for neurobiologists both from a research point of view and from a technical point of view.
Preferably the immortalising agent is, what is typically referred to as, a soft oncogene such as a SV40 viral oncogene and more preferably, in the instance where a control means is preferred the oncogene is the SV40 T antigen which is permissive, that is to say the viral gene active product is expressed, at 33xc2x0 C. and non-permissive, that is to say the viral gene active product is not expressed, at 39xc2x0 C., thus cells immortalised using this agent are temperature sensitive for apoptosis.
Uniquely, our cells, when transformed using SV40 T antigen and exposed to an environment, natural or artificial, which promotes differentiation, survived crisisxe2x80x94a condition which is typically followed by apoptosis.
It would seem that the said environment also provides for the release of substances or somehow effects the cells to enable them to survive apoptosis.
In yet a further preferred embodiment of the invention said cell-line includes a safety feature which allows for selective disabling or destruction of said cell-line. This safety feature is of advantage where the cell-line is to be used for the purpose of transplantation or is otherwise, whether it be permanent or temporary, attached to, administered to, or stored in, an individual. This safety feature allows the cell-line to be selectively disabled, and by this we mean rendered harmless, or destroyed, in instances where the cell-line is thought likely to, or is shown to, have the potential to become tumorigenic in vivo, or is thought to be in any way harmful to an individual.
Our copending patent application GB 9422236.1 teaches how a vector can be produced which provides for co-expression of a safety feature in the form of a gene which may or may not be linked to the immortalising oncogene.
According to a further aspect of the invention there is provided cells and/or cell-lines produced in accordance with the method of the invention. Accordingly there is provided at least one homogeneous population of immortalised cells which can be made to fully differentiate so as to provide a homogeneous population of fully differentiated nerve cells; and/or alternatively, there is provided at least one homogeneous population of immortalised cells provided with means to terminate immortalisation and activate apoptosis.
According to a yet further aspect of the invention there is provided cells and/or cell-lines produced in accordance with a method of the invention which, when differentiated, retain their phenotypic characteristics and/or are non-mitotic and/or survive at low densities.
An embodiment of the invention will now be described by way of example only and for the purpose of example only with specific reference to serotonin secreting functional nerve cells.